Autoimmunity in motion: Mechanisms of immune regulation and destruction revealed by in vivo imaging.

Autoimmunity arises when mechanisms of immune tolerance fail. Here we discuss mechanisms of T cell activation and tolerance and the dynamics of the autoimmune response at the site of disease. Live imaging of autoimmunity provides the ability to analyze immune cell dynamics at the single-cell level within the complex intact environment where disease occurs. These analyses have revealed mechanisms of T cell activation and tolerance in the lymph nodes, mechanisms of T cell entry into sites of autoimmune disease, and mechanisms leading to pathogenesis or protection in the autoimmune lesions. The overarching conclusions point to stable versus transient T cell antigen presenting cell interactions dictating the balance between T cell activation and tolerance, and T cell restimulation as a driver of pathogenesis at the site of autoimmunity. Findings from models of multiple sclerosis and type 1 diabetes are highlighted, however, the results have implications for basic mechanisms of T cell regulation during immune responses, tumor immunity, and autoimmunity.

Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.

During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA-deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA-dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.

Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling.

Adaptive angiogenesis is necessary for tissue repair, however, it may also be associated with the exacerbation of injury and development of chronic disease. In these studies, we demonstrate that lung mesenchymal vascular progenitor cells (MVPC) modulate adaptive angiogenesis via lineage trace, depletion of MVPC, and modulation of ß-catenin expression. Single cell sequencing confirmed MVPC as multipotential vascular progenitors, thus, genetic depletion resulted in alveolar simplification with reduced adaptive angiogenesis. Following vascular endothelial injury, Wnt activation in MVPC was sufficient to elicit an emphysema-like phenotype characterized by increased MLI, fibrosis, and MVPC driven adaptive angiogenesis. Lastly, activation of Wnt/ß-catenin signaling skewed the profile of human and murine MVPC toward an adaptive phenotype. These data suggest that lung MVPC drive angiogenesis in response to injury and regulate the microvascular niche as well as subsequent distal lung tissue architecture via Wnt signaling.

Ena/VASP proteins regulate activated T-cell trafficking by promoting diapedesis during transendothelial migration.

Vasodilator-stimulated phosphoprotein (VASP) and Ena-VASP-like (EVL) are cytoskeletal effector proteins implicated in regulating cell morphology, adhesion, and migration in various cell types. However, the role of these proteins in T-cell motility, adhesion, and in vivo trafficking remains poorly understood. This study identifies a specific role for EVL and VASP in T-cell diapedesis and trafficking. We demonstrate that EVL and VASP are selectively required for activated T-cell trafficking but are not required for normal T-cell development or for naïve T-cell trafficking to lymph nodes and spleen. Using a model of multiple sclerosis, we show an impairment in trafficking of EVL/VASP-deficient activated T cells to the inflamed central nervous system of mice with experimental autoimmune encephalomyelitis. Additionally, we found a defect in trafficking of EVL/VASP double-knockout (dKO) T cells to the inflamed skin and secondary lymphoid organs. Deletion of EVL and VASP resulted in the impairment in α4 integrin (CD49d) expression and function. Unexpectedly, EVL/VASP dKO T cells did not exhibit alterations in shear-resistant adhesion to, or in crawling on, primary endothelial cells under physiologic shear forces. Instead, deletion of EVL and VASP impaired T-cell diapedesis. Furthermore, T-cell diapedesis became equivalent between control and EVL/VASP dKO T cells upon α4 integrin blockade. Overall, EVL and VASP selectively mediate activated T-cell trafficking by promoting the diapedesis step of transendothelial migration in a α4 integrin-dependent manner.

Down regulation of macrophage IFNGR1 exacerbates systemic L. monocytogenes infection.

Interferons (IFNs) target macrophages to regulate inflammation and resistance to microbial infections. The type II IFN (IFNγ) acts on a cell surface receptor (IFNGR) to promote gene expression that enhance macrophage inflammatory and anti-microbial activity. Type I IFNs can dampen macrophage responsiveness to IFNγ and are associated with increased susceptibility to numerous bacterial infections. The precise mechanisms responsible for these effects remain unclear. Type I IFNs silence macrophage ifngr1 transcription and thus reduce cell surface expression of IFNGR1. To test how these events might impact macrophage activation and host resistance during bacterial infection, we developed transgenic mice that express a functional FLAG-tagged IFNGR1 (fGR1) driven by a macrophage-specific promoter. Macrophages from fGR1 mice expressed physiologic levels of cell surface IFNGR1 at steady state and responded equivalently to WT C57Bl/6 macrophages when treated with IFNγ alone. However, fGR1 macrophages retained cell surface IFNGR1 and showed enhanced responsiveness to IFNγ in the presence of type I IFNs. When fGR1 mice were infected with the bacterium Listeria monocytogenes their resistance was significantly increased, despite normal type I and II IFN production. Enhanced resistance was dependent on IFNγ and associated with increased macrophage activation and antimicrobial function. These results argue that down regulation of myeloid cell IFNGR1 is an important mechanism by which type I IFNs suppress inflammatory and anti-bacterial functions of macrophages.

Equity trade-offs in conservation decision making.

Conservation decisions increasingly involve multiple environmental and social objectives, which result in complex decision contexts with high potential for trade-offs. Improving social equity is one such objective that is often considered an enabler of successful outcomes and a virtuous ideal in itself. Despite its idealized importance in conservation policy, social equity is often highly simplified or ill-defined and is applied uncritically. What constitutes equitable outcomes and processes is highly normative and subject to ethical deliberation. Different ethical frameworks may lead to different conceptions of equity through alternative perspectives of what is good or right. This can lead to different and potentially conflicting equity objectives in practice. We promote a more transparent, nuanced, and pluralistic conceptualization of equity in conservation decision making that particularly recognizes where multidimensional equity objectives may conflict. To help identify and mitigate ethical conflicts and avoid cases of good intentions producing bad outcomes, we encourage a more analytical incorporation of equity into conservation decision making particularly during mechanistic integration of equity objectives. We recommend that in conservation planning motivations and objectives for equity be made explicit within the problem context, methods used to incorporate equity objectives be applied with respect to stated objectives, and, should objectives dictate, evaluation of equity outcomes and adaptation of strategies be employed during policy implementation.

Crowdfunding biodiversity conservation.

Raising funds is critical for conserving biodiversity and hence so is scrutinizing emerging financial mechanisms that may help achieve this goal. Anecdotal evidence indicates crowdfunding is being used to support activities needed for biodiversity conservation, yet its magnitude and allocation remain largely unknown. To help address this knowledge gap, we conducted a global analysis based on conservation-focused projects extracted from crowdfunding platforms. For each project, we determined the funds raised, date, country of implementation, proponent characteristics, activity type, biodiversity realm, and target taxa. We identified 72 relevant platforms and 577 conservation-focused projects that raised $4,790,634 since 2009. Although proponents were based in 38 countries, projects were delivered across 80 countries, indicating a potential mechanism of resource mobilization. Proponents were affiliated with nongovernmental organizations (35%) or universities (30%) or were freelancers (26%). Most projects were for research (40%), persuasion (31%), and on-the-ground actions (21%). Projects were more focused on species (57.7%) and terrestrial ecosystems (20.3%), and less focused on marine (8.8%) and freshwater ecosystems (3.6%). Projects focused on 208 species, including a disproportionate number of threatened birds and mammals. Crowdfunding for biodiversity conservation is a global phenomenon and there is potential for expansion, despite possible pitfalls (e.g., uncertainty about effectiveness). Opportunities to advance conservation through crowdfunding arise from its capacity to mobilize funds spatially and increase steadily over time, inclusion of overlooked species, adoption by multiple actors, and funding of activities beyond research. Our findings pave the way for further research on key questions, such as campaign success rates, effectiveness of conservation actions, and drivers of crowdfunding adoption. Even though crowdfunding capital raised has been modest relative to other conservation-finance mechanisms, its contribution goes beyond funding research and providing capital. Embraced with due care, crowdfunding could become an important financial mechanism for biodiversity conservation.

Cutting Edge: Nonobese Diabetic Mice Deficient in Chromogranin A Are Protected from Autoimmune Diabetes.

T cells reactive to ß cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the ß cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA(+/+) mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

A synaptic basis for paracrine interleukin-2 signaling during homotypic T cell interaction.

T cells slow their motility, increase adherence, and arrest after encounters with antigen-presenting cells (APCs) bearing peptide-MHC complexes. Here, we analyzed the cell-cell communication among activating T cells. In vivo and in vitro, activating T cells associated in large clusters that collectively persisted for >30 min, but they also engaged in more transient interactions, apparently distal to APCs. Homotypic aggregation was driven by LFA-1 integrin interactions. Ultrastructural analysis revealed that cell-cell contacts between activating T cells were organized as multifocal synapses, and T cells oriented both the microtubule-organizing complex and interleukin-2 (IL-2) secretion toward this synapse. T cells engaged in homotypic interactions more effectively captured IL-2 relative to free cells. T cells receiving paracrine synaptic IL-2 polarized their IL-2 signaling subunits into the synaptic region and more efficiently phosphorylated the transcription factor STAT5, likely through a synapse-associated signaling complex. Thus, synapse-mediated cytokine delivery accelerates responses in activating T cells.

CD11c-Expressing B Cells Are Located at the T Cell/B Cell Border in Spleen and Are Potent APCs.

In addition to the secretion of Ag-specific Abs, B cells may play an important role in the generation of immune responses by efficiently presenting Ag to T cells. We and other investigators recently described a subpopulation of CD11c(+) B cells (Age/autoimmune-associated B cells [ABCs]) that appear with age, during virus infections, and at the onset of some autoimmune diseases and participate in autoimmune responses by secreting autoantibodies. In this study, we assessed the ability of these cells to present Ag and activate Ag-specific T cells. We demonstrated that ABCs present Ag to T cells, in vitro and in vivo, better than do follicular B cells (FO cells). Our data indicate that ABCs express higher levels of the chemokine receptor CCR7, have higher responsiveness to CCL21 and CCL19 than do FO cells, and are localized at the T/B cell border in spleen. Using multiphoton microscopy, we show that, in vivo, CD11c(+) B cells form significantly more stable interactions with T cells than do FO cells. Together, these data identify a previously undescribed role for ABCs as potent APCs and suggest another potential mechanism by which these cells can influence immune responses and/or the development of autoimmunity.

Antigen recognition in the islets changes with progression of autoimmune islet infiltration.

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention because immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration, we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune NOD mice. Using both intravital and explanted two-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was Ag dependent and due, at least in part, to Ag recognition through sustained interactions with CD11c(+) APCs. As islet infiltration progressed, T cell motility became Ag independent, with a loss of T cell arrest and sustained interactions with CD11c(+) APCs. These studies suggest that the autoimmune T cell response in the islets may be temporarily dampened during the course of islet infiltration and disease progression.

An evolving autoimmune microenvironment regulates the quality of effector T cell restimulation and function.

Defining the processes of autoimmune attack of tissues is important for inhibiting continued tissue destruction. In type 1 diabetes, it is not known how cytotoxic effector T cell responses evolve over time in the pancreatic islets targeted for destruction. We used two-photon microscopy of live, intact, individual islets to investigate how progression of islet infiltration altered the behavior of infiltrating islet-specific CD8(+) T cells. During early-islet infiltration, T-cell interactions with CD11c(+) antigen-presenting cells (APCs) were stable and real-time imaging of T cell receptor (TCR) clustering provided evidence of TCR recognition in these stable contacts. Early T cell-APC encounters supported production of IFN-γ by T effectors, and T cells at this stage also killed islet APCs. At later stages of infiltration, T-cell motility accelerated, and cytokine production was lost despite the presence of higher numbers of infiltrating APCs that were able to trigger T-cell signaling in vitro. Using timed introduction of effector T cells, we demonstrate that elements of the autoimmune-tissue microenvironment control the dynamics of autoantigen recognition by T cells and their resulting pathogenic effector functions.

Evolving immune circuits are generated by flexible, motile, and sequential immunological synapses.

The immune system is made up of a diverse collection of cells, each of which has distinct sets of triggers that elicit unique and overlapping responses. It is correctly described as a 'system' because its overall properties (e.g. 'tolerance', 'allergy') emerge from multiple interactions of its components cells. To mobilize a response where needed, the majority of the cells of the system are obligatorily highly motile and so must communicate with one another over both time and space. Here, we discuss the flexibility of the primary immunological synapse (IS) with respect to motility. We then consider the primary IS as an initiating module that licenses 'immunological circuits': the latter consisting of two or more cell-cell synaptic interactions. We discuss how two or three component immunological circuits interact might with one another in sequence and how the timing, stoichiometry, milieu, and duration of assembly of immunological circuits are likely to be key determinants in the emergent outcome and thus the system-wide immune response. An evolving consideration of immunological circuits, with an emphasis on the cell-cell modules that complement T-antigen-presenting cell interaction, provides a fundamental starting point for systems analysis of the immune response.

Host DNA released in response to aluminum adjuvant enhances MHC class II-mediated antigen presentation and prolongs CD4 T-cell interactions with dendritic cells.

Many vaccines include aluminum salts (alum) as adjuvants despite little knowledge of alum's functions. Host DNA rapidly coats injected alum. Here, we further investigated the mechanism of alum and DNA's adjuvant function. Our data show that DNase coinjection reduces CD4 T-cell priming by i.m. injected antigen + alum. This effect is partially replicated in mice lacking stimulator of IFN genes, a mediator of cellular responses to cytoplasmic DNA. Others have shown that DNase treatment impairs dendritic cell (DC) migration from the peritoneal cavity to the draining lymph node in mice immunized i.p. with alum. However, our data show that DNase does not affect accumulation of, or expression of costimulatory proteins on, antigen-loaded DCs in lymph nodes draining injected muscles, the site by which most human vaccines are administered. DNase does inhibit prolonged T-cell-DC conjugate formation and antigen presentation between antigen-positive DCs and antigen-specific CD4 T cells following i.m. injection. Thus, from the muscle, an immunization site that does not require host DNA to promote migration of inflammatory DCs, alum acts as an adjuvant by introducing host DNA into the cytoplasm of antigen-bearing DCs, where it engages receptors that promote MHC class II presentation and better DC-T-cell interactions.

CB2 receptor deficiency increases amyloid pathology and alters tau processing in a transgenic mouse model of Alzheimer's disease.

The endocannabinoid CB2 receptor system has been implicated in the neuropathology of Alzheimer's disease (AD). In order to investigate the impact of the CB2 receptor system on AD pathology, a colony of mice with a deleted CB2 receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB2 receptor-sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2(-/-) (Cnr2(tm1Dgen)/J) mice to produce a colony of J20 CNR2(+/+) and J20 CNR2(-/-) mice. Seventeen J20 CNR2(+/+) mice (12 females, 5 males) and 16 J20 CNR2(-/-) mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aß production and plaque deposition quantified with 6E10 staining, the effect of CB2 receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aß42 and plaque deposition were significantly increased in J20 CNR2(-/-) mice relative to CNR2(+/+) mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2(-/-) mice. Total tau was significantly suppressed in J20 CNR2(-/-) mice relative to J20 CNR2(+/+) mice. The results confirm the constitutive role of the CB2 receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB2 to reduce Aß; however, the results suggest that interventions may have a divergent effect on tau pathology.

Network approach reveals preferential T-cell and macrophage association with α-linked ß-cells in early stage of insulitis in NOD mice.

One of the challenges in studying islet inflammation - insulitis - is that it is a transient phenomenon. Traditional reporting of the insulitis progression is based on cumulative, donor-averaged values of leucocyte density in the vicinity of pancreatic islets, that hinders intra- and inter-islet heterogeneity of disease progression. Here, we aimed to understand why insulitis is non-uniform, often with peri-insulitis lesions formed on one side of an islet. To achieve this, we demonstrated applicability of network theory in detangling intra-islet multi-cellular interactions during insulitis. Specifically, we asked the question "what is unique about regions of the islet which interact with immune cells first". This study utilized the non-obese diabetic mouse model of type one diabetes and examined the interplay among α-, ß-, T-cells, myeloid cells, and macrophages in pancreatic islets during the progression of insulitis. Disease evolution was tracked based on T/ß cell ratio in individual islets. In the early stage, we found that immune cells are preferentially interacting with α-cell-rich regions of an islet. At the islet periphery α-linked ß-cells were found to be targeted significantly more compared to those without α-cell neighbors. Additionally, network analysis revealed increased T-myeloid, and T-macrophage interactions with all ß-cells.

CB2 receptor deficiency increases amyloid pathology and alters tau processing in a transgenic mouse model of Alzheimer's disease.

The endocannabinoid CB2 receptor system has been implicated in the neuropathology of Alzheimer's disease (AD). In order to investigate the impact of the CB2 receptor system on AD pathology, a colony of mice with a deleted CB2 receptor gene, CNR2, was established on a transgenic human mutant APP background for pathological comparison with CB2 receptor-sufficient transgenic mice. J20 APP (PDGFB-APPSwInd) mice were bred over two generations with CNR2⁻/⁻ (Cnr2(tm1Dgen)/J) mice to produce a colony of J20 CNR2⁺/⁺ and J20 CNR2⁻/⁻ mice. Seventeen J20 CNR2⁺/⁺ mice (12 females, 5 males) and 16 J20 CNR2⁻/⁻ mice (11 females, 5 males) were killed at 12 months, and their brains were interrogated for AD-related pathology with both biochemistry and immunocytochemistry (ICC). In addition to amyloid-dependent endpoints such as soluble Aß production and plaque deposition quantified with 6E10 staining, the effect of CB2 receptor deletion on total soluble mouse tau production was assayed by using a recently developed high-sensitivity assay. Results revealed that soluble Aß42 and plaque deposition were significantly increased in J20 CNR2⁻/⁻ mice relative to CNR2⁺/⁺ mice. Microgliosis, quantified with ionized calcium-binding adapter molecule 1 (Iba-1) staining, did not differ between groups, whereas plaque associated microglia was more abundant in J20 CNR2⁻/⁻ mice. Total tau was significantly suppressed in J20 CNR2⁻/⁻ mice relative to J20 CNR2⁺/⁺ mice. The results confirm the constitutive role of the CB2 receptor system both in reducing amyloid plaque pathology in AD and also support tehpotential of cannabinoid therapies targeting CB2 to reduce Aß; however, the results suggest that interventions may have a divergent effect on tau pathology.

Duration of antigen receptor signaling determines T-cell tolerance or activation.

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells, we show, using single-cell biochemical analyses, that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction, as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance, and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation.